Synthetic oligonucleotides designed to affect HIV pathogenesis by sequence-specific interactions with viral nucleic acid have shown promise as candidates for antiviral therapy. We have been exploring the comparative efficacy of oligonucleotide treatment (aimed at different stages in the viral replicative cycle) of HIV-1 infected cells in vitro. The family of oligos we have used target the same polypurine-rich sequences, but carry different modifications such that they may function as triple helix (TH), antisense (AS), or nonspecific inhibitors. Treatment of infected cells with ONs designed to form a Triple Helix (TH) with the target HIV sequence significantly decreased p24 antigen levels as well as decreasing the amounts of viral DNA and RNA relative to untreated cells. Treatment of infected cells with AS ONs also inhibited HIV expression, but in a more limited and less sequence specific manner. The oligos were rapidly taken up by the cells, entering the nuclei within 10 minutes and maintaining detectable, non-degraded levels for up to 48 hours. These oligos showed some interaction with their target sequences in in vitro assays, but the association was not stable. We have continued these studies with TH oligos carrying different modifications designed to 1) increase the stability of the TH structure, 2) provide more resistance to cellular nucleases, and 3) enhance the antiviral activity of the ON - target interaction. We have also begun studies to investigate the primary mode of action in ON inhibition of viral replication. These studies have been conducted in tissue culture cell lines (H9 cells) and with primary normal human peripheral blood lymphocytes (PBLs), using laboratory and primary isolates.